Background: Prostate cancer incidence and death rates are among the highest in American men. The etiology of prostate cancer is not known but based on the literature the catechol-O-methyltransferase (COMT) gene is capable of blocking the carcinogenic estrogen metabolic pathway by metabolizing catechol-estrogens rendering them inactive and thus, plays a role in preventing carcinogenesis. Preliminary data and prior publications from the laboratory support a protective role for COMT in prostate cancer. Research Objectives: The main goal of this project is to determine the role of the COMT gene in prostate cancer. We hypothesize that: (1) reduced COMT is a risk factor for prostate cancer, progression and poor patient outcome, (2) over-expressing the COMT gene can inhibit prostate cancer cells by affecting cell growth and progression pathways, and (3) COMT expression in prostate cells is regulated by epigenetic factors. Project Design and Methods: Aim #1. COMT expression in prostate cancer patients. Existing resources of prostate tissues consisting of benign prostatic hyperplasia and matched normal and cancer specimens from cancer patients will be utilized. The following experiments will be performed: (a) microdissection of normal adjacent and cancer regions from prostate cancer tissues, (b) determine COMT RNA expression levels by real-time PCR, (c) determine COMT protein levels by immunohistochemistry, and (d) determine if COMT expression is associated with cancer incidence, stage, grade, and patient outcome. Accomplishment of these goals will determine whether COMT down-regulation is associated with prostate cancer and assess COMT as a biomarker for prostate cancer. Aim #2. Functional significance of COMT in prostate cancer cells. Prostate cell lines will be used for the following experiments: (a) analyze constitutive COMT expression levels in normal and prostate cancer (androgen dependent and independent or castration resistant) cells by real-time PCR and Western blot analyses, (b) over-express COMT in those cancer cell lines with low COMT expression by establishing stable COMT-expressing clones, (c) measure methoxyestrogen level, cell proliferation, cell cycle distribution, apoptosis, cell invasion, and migration in COMT-expressing cells by flow cytometry and biological assays, (d) evaluate the effects of COMT on cell morphology by microscopy and expression of other genes involved in cell proliferation and cancer progression pathways by gene arrays and Western blot analyses, and (e) determine the effect of COMT on prostate tumor growth in vivo. Accomplishment of these experiments will determine the functional effects of COMT on cell growth and progression pathways in prostate cancer which may implicate COMT as a potential therapeutic target for prostate cancer treatment. Aim #3. Epigenetic regulation of the COMT gene in prostate cancer. Clinical patient specimens (Aim #1) and cell lines (Aim #2) will be used in the following experiments: (a) analyze COMT DNA methylation using sodium bisulfite modification / methylation-specific PCR, (b) determine the histone modification status using chromatin- immunoprecipitation analysis, (c) measure the activity of histone modifying enzymes in cells, (d) correlate COMT epigenetic status and enzyme activities with RNA expression levels determined in Aims #1 and #2, and (e) Determine if demethylating agents and histone deacetylase inhibitors can re-express COMT in prostate cancer cells. Accomplishment of these experiments will determine the mechanisms of regulation of COMT expression in prostate cancer. Veterans Clinical Relevance: The proposed research will generate important insights into COMT's role in prostate cancer etiology, progression, and patient outcome. This could lead to improved methods of identifying Veterans at higher risk for prostate cancer. The proposed research may also reveal COMT to be an important biomarker and therapeutic target for prostate cancer that could translate to the clinic and benefit Veterans.